Although it is generally understood that the amino acid chains that comprise peptides in solution are highly dynamic, one does not expect a change in the primary structure (i.e.
, amino acid sequence) of these molecules to occur. However, in the gas phase, where it is possible to add substantial internal energy, the fragments of these linear structures can undergo rearrangement processes in which the sequence appears to change.1,2
This is especially true when peptide ions are subjected to collisions with a background gas to induce collision-induced dissociation (CID). Generally, the masses of the CID product ions are expected to reflect the amino acid sequence of the peptide and therefore be valuable for sequencing.3,4
In fact, this approach is now a cornerstone in the identification of proteins by mass spectrometry.5,6
The interpretation of these spectra is often carried out using computer algorithms.7,8
With current methods a large fraction of examined ions cannot be identified.9
At least in part, this is due to an incomplete understanding of the underlying fragmentation chemistry.10
As background, it is well-known that under CID conditions, protonated peptide ions generally display abundant cleavages at the backbone amide bonds, giving rise to N-terminal b
and C-terminal y
In proposed mechanisms, the ionizing proton is thought to be moved from a site of higher proton affinity, such as a basic side chain (e.g.
, guanidine group of arginine), to the backbone C=O and then NH groups, thereby weakening the amide bond.12,13
Although there has been controversy about the dissociation mechanism and the types of structures that are formed (especially for b
it is now generally accepted that the most likely dissociation pathway occurs via nucleophilic attack from a backbone carbonyl oxygen to a carbonyl carbon, thus forming a five-membered “oxazolone” ring b
This is exemplified in the reaction chemistry of the pentapeptide Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) that we have chosen in this study (see ). Detailed ab inito
calculations have confirmed that this pathway is both energetically and entropically accessible.10
Moreover, the presence of oxazolone-type structures has recently been corroborated on the basis of structurally diagnostic oxazolone C=O stretch modes by infrared (IR) spectroscopy.19,20
Linear oxazolone structures are believed to be able to isomerize to fully cyclic structures, following a nucleophilic attack by the N-terminus on the oxazolone ring ().2
If the cyclic structure opens up at a different amide bond than where it was originally formed, this results in oxazolone structures with permutated (i.e.
, scrambled) primary structures (gray box, ). The subsequent CID of these scrambled structures is responsible for the appearance of “nondirect” CID fragments,2
which cannot be explained on the basis of the original amino acid sequence of the peptide.
Reaction Scheme for Generating b4 and a4 CID Product Ions from the Protonated Leu-Enkephalin Pentapeptide Precursor Iona
Experimental evidence for such cyclic structures comes from IR spectroscopy; however, the assignment is less definite than in the case of oxazolone structures, and it is difficult to draw conclusions on the relative abundances of the different isomers in the mixture.20
Other indicative evidence for cyclic structures comes from H/D exchange studies21
and ion mobility measurements.22
It should be noted that cyclic configurations are not limited to b
ions but can also occur for other N-terminal fragments, such as for instance a
, the b
ion less a CO). In analogy to b4
, Leu-enkephalin a4
is thought to adopt both linear imine (E) and cyclic structures (F) ().20
A re-opening of the cyclic isomers can again give rise to permutated oxazolone-type structures (gray box G), the subsequent CID of which rationalizes nondirect sequence ions.
Here, we present a systematic study of the structural products that are formed in collision-induced dissociation of the pentapeptide Leu-enkephalin by ion mobility/mass spectrometry.23,24
These products are also compared to the fragments for N
-acetyl Leu-enkephalin. We address the question of the relative abundances of the different structural isomers, and whether the relative abundances can be influenced by the amount of energy deposited in the molecule. This result provides insights into the dynamics of the isomerization pathways, which are important in peptide scrambling processes. This is also the first study that uses ion mobility in combination with theoretical approaches to answer structural questions on CID fragment ions.